Standoff Deep Ultraviolet Raman Spectrometer for Trace Detection.

We developed a state-of-the-art, high-sensitivity, low-stray-light standoff deep-ultraviolet (DUV) Raman spectrometer for the trace detection of resonance Raman-enhanced chemical species. As an excitation source for Raman measurements, we utilized our recently developed, second-generation, miniaturized, diode-pumped, solid-state neodymium-doped gadolinium orthovanadate (Nd:GdVO4) laser that generates quasi-continuous wave 228 nm light. This 228 nm excitation enhances the Raman intensities of vibrations of NOx groups in explosive molecules, aromatic groups in biological molecules, and various aromatic hydrocarbons. Our DUV Raman spectrograph utilizes a custom DUV f/8 Cassegrain telescope with an ∼200 mm diameter primary mirror, high-efficiency DUV transmission gratings, custom DUV mirrors, and a custom 228 nm Rayleigh rejection filter. We utilized our new standoff DUV Raman spectrometer to measure high signal-to-noise ratio spectra of ∼50 µg/cm2 drop-cast explosives: ammonium nitrate (AN), trinitrotoluene, pentaerythritol tetranitrate as well as aromatic biological molecules: lysozyme, tryptophan, tyrosine, deoxycytidine monophosphate, deoxyadenosine monophosphate at an ∼3 m distance within 10-30 s accumulation times. We roughly estimate the average ultraviolet resonance Raman (UVRR) detection limits for the relatively homogeneous drop-cast films of explosives and biological molecules to be ∼1 µg/cm2 when utilizing a continuous raster scanning that averages Raman signal over ∼1 cm2 sample area to avoid quick analyte depletion due to ultraviolet (UV) photolysis. We determined 3 m standoff UVRR detection limits for drop-cast AN films and identified factors impacting UVRR detection limits such as analyte photochemistry and analyte morphology. We found a detection limit of ∼0.5 µg/cm2 for drop-cast AN films on glass substrates when the Raman signal is averaged over ∼0.5 cm2 of sample surface using a continuous raster scan. For a step raster scan, when the probed sample area is limited to the laser spot size, the detection limit is approximately tenfold higher (∼5 µg/cm2) due to the impact of UV photochemistry.

Diverse organic-mineral associations in Jezero crater, Mars.

The presence and distribution of preserved organic matter on the surface of Mars can provide key information about the Martian carbon cycle and the potential of the planet to host life throughout its history. Several types of organic molecules have been previously detected in Martian meteorites1 and at Gale crater, Mars2-4. Evaluating the diversity and detectability of organic matter elsewhere on Mars is important for understanding the extent and diversity of Martian surface processes and the potential availability of carbon sources1,5,6. Here we report the detection of Raman and fluorescence spectra consistent with several species of aromatic organic molecules in the Máaz and Séítah formations within the Crater Floor sequences of Jezero crater, Mars. We report specific fluorescence-mineral associations consistent with many classes of organic molecules occurring in different spatial patterns within these compositionally distinct formations, potentially indicating different fates of carbon across environments. Our findings suggest there may be a diversity of aromatic molecules prevalent on the Martian surface, and these materials persist despite exposure to surface conditions. These potential organic molecules are largely found within minerals linked to aqueous processes, indicating that these processes may have had a key role in organic synthesis, transport or preservation.

Aqueous alteration processes in Jezero crater, Mars-implications for organic geochemistry.

The Perseverance rover landed in Jezero crater, Mars, in February 2021. We used the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument to perform deep-ultraviolet Raman and fluorescence spectroscopy of three rocks within the crater. We identify evidence for two distinct ancient aqueous environments at different times. Reactions with liquid water formed carbonates in an olivine-rich igneous rock. A sulfate-perchlorate mixture is present in the rocks, which probably formed by later modifications of the rocks by brine. Fluorescence signatures consistent with aromatic organic compounds occur throughout these rocks and are preserved in minerals related to both aqueous environments.

Calibration of the SHERLOC Deep Ultraviolet Fluorescence-Raman Spectrometer on the Perseverance Rover.

We describe the wavelength calibration of the spectrometer for the scanning of habitable environments with Raman and luminescence for organics and chemicals (SHERLOC) instrument onboard NASA's Perseverance Rover. SHERLOC utilizes deep ultraviolet Raman and fluorescence (DUV R/F) spectroscopy to enable analysis of samples from the Martian surface. SHERLOC employs a 248.6 nm deep ultraviolet laser to generate Raman-scattered photons and native fluorescence emission photons from near-surface material to detect and classify chemical and mineralogical compositions. The collected photons are focused on a charge-coupled device and the data are returned to Earth for analysis. The compact DUV R/F spectrometer has a spectral range from 249.9 nm to 353.6 nm (∼200 cm-1 to 12 000 cm-1) (with a spectral resolution of 0.296 nm (∼40 cm-1)). The compact spectrometer uses a custom design to project a high-resolution Raman spectrum and a low-resolution fluorescence spectrum on a single charge-coupled device. The natural spectral separation enabled by deep ultraviolet excitation enables wavelength separation of the Raman/fluorescence spectra. The SHERLOC spectrometer was designed to optimize the resolution of the Raman spectral region and the wavelength range of the fluorescence region. The resulting illumination on the charge-coupled device is curved, requiring a segmented, nonlinear wavelength calibration in order to understand the mineralogy and chemistry of Martian materials.

Deep Ultraviolet Standoff Photoacoustic Spectroscopy of Trace Explosives.

We demonstrate deep ultraviolet (UV) photoacoustic spectroscopy (PAS) of trace explosives using a sensitive microphone at meter standoff distances. We directly detect 10 µg/cm2 of pentaerythritol tetranitrate (PETN), 2,4,6-trinitrotoluene (TNT), and ammonium nitrate (AN) with 1 s accumulations from a 3 m standoff distance. Large PAS signals for standoff detection are achieved by exciting into the absorption bands of the explosives with a 213 nm laser. We also investigate the impact of the deep UV photochemistry of AN on the PAS signal strength and stability. We find that production of gaseous species during photolysis of AN enhances the PAS signal strength. This deep UV photochemistry can, however, limit the PAS signal lifetimes when detecting trace quantities.

Hydrophobic Collapse Initiates the Poly( N-isopropylacrylamide) Volume Phase Transition Reaction Coordinate.

The best-known examples of smart, responsive hydrogels derive from poly( N-isopropylacrylamide) (PNIPAM) cross-linked polymer networks. These hydrogels undergo volume phase transitions (VPTs) triggered by temperature, chemical, and/or environmental changes. PNIPAM hydrogels can undergo more than 50-fold volume changes within ∼1 µs intervals. Studies have tried to elucidate the molecular mechanism of these extraordinarily large responses. Nevertheless, the molecular reaction coordinates that drive the VPT remain unclear. Using visible nonresonance Raman temperature-jump spectroscopy, we determined the molecular ordering of this VPT. The PNIPAM hydrophobic isopropyl and methylene groups dehydrate with time constants of 109 ± 64 and 104 ± 44 ns, initiating the volume collapse of PNIPAM. The subsequent dehydration of the PNIPAM amide groups is significantly slower, as our group previously discovered (360 ± 85 ns). This determination of the ordering of the molecular reaction coordinate of the PNIPAM VPT enables the development of the next generation of super-responsive materials.

UV Resonance Raman Investigation of Pentaerythritol Tetranitrate Solution Photochemistry and Photoproduct Hydrolysis.

Ultraviolet resonance Raman spectroscopy (UVRR) is being developed for standoff trace explosives detection. To accomplish this, it is important to develop a deep understanding of the accompanying UV excited photochemistry of explosives, as well as the impact of reactions on the resulting photoproducts. In the work here we used 229 nm excited UVRR spectroscopy to monitor the photochemistry of pentaerythritol tetranitrate (PETN) in acetonitrile. We find that solutions of PETN in CD3CN photodegrade with a quantum yield of 0.08 ± 0.02, as measured by high performance liquid chromatography (HPLC). The initial step in the 229 nm UV photolysis of PETN in CD3CN is cleavage of an O-NO2 bond to form NO2. The accompanying photoproduct is pentaerythritol trinitrate (PETriN), (CH2ONO2)3CCH2OH formed by photolysis of a single O-NO2. The resulting UVRR spectra show a dominant photoproduct band at ∼1308 cm-1, which derives from the symmetric stretch of dissolved NO2. This photoproduct NO2 is hydrolyzed by trace amounts of water, which downshifts this 1308 cm-1 NO2 Raman band due to the formation of molecular HNO3. The dissociation of HNO3 to NO3- in the presence of additional water results in an intense NO3- symmetric stretching UVRR band at 1044 cm-1.

Ultraviolet Raman Wide-Field Hyperspectral Imaging Spectrometer for Standoff Trace Explosive Detection.

We constructed the first deep ultraviolet (UV) Raman standoff wide-field imaging spectrometer. Our novel deep UV imaging spectrometer utilizes a photonic crystal to select Raman spectral regions for detection. The photonic crystal is composed of highly charged, monodisperse 35.5 ± 2.9 nm silica nanoparticles that self-assemble in solution to produce a face centered cubic crystalline colloidal array that Bragg diffracts a narrow ∼1.0 nm full width at half-maximum (FWHM) UV spectral region. We utilize this photonic crystal to select and image two different spectral regions containing resonance Raman bands of pentaerythritol tetranitrate (PETN) and NH4NO3 (AN). These two deep UV Raman spectral regions diffracted were selected by angle tuning the photonic crystal. We utilized this imaging spectrometer to measure 229 nm excited UV Raman images containing ∼10-1000 µg/cm2 samples of solid PETN and AN on aluminum surfaces at 2.3 m standoff distances. We estimate detection limits of ∼1 µg/cm2 for PETN and AN films under these experimental conditions.

Aluminum Film-Over-Nanosphere Substrates for Deep-UV Surface-Enhanced Resonance Raman Spectroscopy.

We report here the first fabrication of aluminum film-over nanosphere (AlFON) substrates for UV surface-enhanced resonance Raman scattering (UVSERRS) at the deepest UV wavelength used to date (λex = 229 nm). We characterize the AlFONs fabricated with two different support microsphere sizes using localized surface plasmon resonance spectroscopy, electron microscopy, SERRS of adenine, tris(bipyridine)ruthenium(II), and trans-1,2-bis(4-pyridyl)-ethylene, SERS of 6-mercapto-1-hexanol (as a nonresonant molecule), and dielectric function analysis. We find that AlFONs fabricated with the 210 nm microspheres generate an enhancement factor of approximately 104-5, which combined with resonance enhancement of the adsorbates provides enhancement factors greater than 106. These experimental results are supported by theoretical analysis of the dielectric function. Hence our results demonstrate the advantages of using AlFON substrates for deep UVSERRS enhancement and contribute to broadening the SERS application range with tunable and affordable substrates.

Compact Solid-State 213 nm Laser Enables Standoff Deep Ultraviolet Raman Spectrometer: Measurements of Nitrate Photochemistry.

We describe a new compact acousto-optically Q-switched diode-pumped solid-state (DPSS) intracavity frequency-tripled neodymium-doped yttrium vanadate laser capable of producing ~100 mW of 213 nm power quasi-continuous wave as 15 ns pulses at a 30 kHz repetition rate. We use this new laser in a prototype of a deep ultraviolet (UV) Raman standoff spectrometer. We use a novel high-throughput, high-resolution Echelle Raman spectrograph. We measure the deep UV resonance Raman (UVRR) spectra of solid and solution sodium nitrate (NaNO3) and ammonium nitrate (NH4NO3) at a standoff distance of ~2.2 m. For this 2.2 m standoff distance and a 1 min spectral accumulation time, where we only monitor the symmetric stretching band, we find a solid state NaNO3 detection limit of ~100 µg/cm(2). We easily detect ~20 µM nitrate water solutions in 1 cm path length cells. As expected, the aqueous solutions UVRR spectra of NaNO3 and NH4NO3 are similar, showing selective resonance enhancement of the nitrate (NO3(-)) vibrations. The aqueous solution photochemistry is also similar, showing facile conversion of NO3(-) to nitrite (NO2(-)). In contrast, the observed UVRR spectra of NaNO3 and NH4NO3 powders significantly differ, because their solid-state photochemistries differ. Whereas solid NaNO3 photoconverts with a very low quantum yield to NaNO2, the NH4NO3 degrades with an apparent quantum yield of ~0.2 to gaseous species.

Solution and Solid Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Ultraviolet (UV) 229 nm Photochemistry.

We measured the 229 nm deep-ultraviolet resonance Raman (DUVRR) spectra of solution and solid-state hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also examined the photochemistry of RDX both in solution and solid states. RDX quickly photodegrades with a solution quantum yield of φ ~ 0.35 as measured by high-performance liquid chromatography (HPLC). New spectral features form over time during the photolysis of RDX, indicating photoproduct formation. The photoproduct(s) show stable DUVRR spectra at later irradiation times that allow standoff detection. In the solution-state photolysis, nitrate is a photoproduct that can be used as a signature for detection of RDX even after photolysis. We used high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and gas chromatography mass spectrometry (GCMS) to determine some of the major solution-state photoproducts. X-ray photoelectron spectroscopy (XPS) was also used to determine photoproducts formed during solid-state RDX photolysis.

Raman hyperspectral imaging spectrometer utilizing crystalline colloidal array photonic crystal diffraction.

We fabricated a novel hyperspectral Raman imaging spectrometer that, for the first time, uses a photonic-crystal wavelength-selecting device to select a narrow-wavelength spectral interval. The photonic crystal consists of an array of highly charged, monodisperse polystyrene particles that self-assemble into a face-centered cubic crystal. The photonic crystal Bragg-diffracts a narrow spectral interval that can be tuned by altering the incident angle of collimated Raman scattered light. Our prototype spectrometer diffracts a ~200 cm(-1) interval of the 488 nm excited visible Raman spectrum of Teflon. This enabled us to select a close-lying triplet of Teflon Raman bands. We imaged the Teflon surface by focusing this narrow region onto a charge-coupled device to create a Raman image of the sample surface that spectrally details the chemical composition.

Solution and solid trinitrotoluene (TNT) photochemistry: persistence of TNT-like ultraviolet (UV) resonance Raman bands.

We examined the 229 nm deep-ultraviolet resonance Raman (DUVRR) spectra of solution and solid-state trinitrotoluene (TNT) and its solution and solid-state photochemistry. Although TNT photodegrades with a solution quantum yield of ϕ ∼ 0.015, the initial photoproducts show DUVRR spectra extraordinarily similar to pure TNT, due to the similar photoproduct enhancement of the -NO2 stretching vibrations. This results in TNT-like DUVRR spectra even after complete TNT photolysis. These ultraviolet resonance Raman spectral bands enable DUVRR of trace as well as DUVRR standoff TNT detection. We determined the structure of various initial TNT photoproducts by using liquid chromatography-mass spectrometry and tandem mass spectrometry. Similar TNT DUVRR spectra and photoproducts are observed in the solution and solid states.

High-throughput, high-resolution Echelle deep-UV Raman spectrometer.

We constructed an ultrahigh-throughput, high-resolution ultraviolet (UV) Raman spectrograph that utilizes a high-efficiency filter-stage monochromator and a high-dispersion Echelle spectrograph. The spectrograph utilizes a total of six mirrors and two gratings, with an overall efficiency at 229 nm of ~18%. The limiting resolution of our spectrometer is 0.6 cm⁻¹ full width half-maximum (FWHM), as measured for 229 nm Rayleigh scattering. Use of a 1 mm-wide entrance slit gives rise to an approximately 10 cm⁻¹ FWHM resolution at 229 nm. The ultrahigh spectrograph throughput enables ultrahigh signal-to-noise ratio, deep UV Raman spectra that allow us to monitor <1% changes in peptide bond composition. The throughput is measured to be 35-fold greater than conventional deep UV Raman spectrometers.

UV Resonance Raman Elucidation of the Terminal and Internal Peptide Bond Conformations of Crystalline and Solution Oligoglycines.

Spectroscopic investigations of macromolecules generally attempt to interpret the measured spectra in terms of the summed contributions of the different molecular fragments. This is the basis of the local mode approximation in vibrational spectroscopy. In the case of resonance Raman spectroscopy independent contributions of molecular fragments require both a local mode-like behavior and the uncoupled electronic transitions. Here we show that the deep UV resonance Raman spectra of aqueous solution phase oligoglycines show independent peptide bond molecular fragment contributions indicating that peptide bonds electronic transitions and vibrational modes are uncoupled. We utilize this result to separately determine the conformational distributions of the internal and penultimate peptide bonds of oligoglycines. Our data indicate that in aqueous solution the oligoglycine terminal residues populate conformations similar to those found in crystals (3(1)-helices and ß-strands), but with a broader distribution, while the internal peptide bond conformations are centered around the 3(1)-helix Ramachandran angles.

UV resonance raman investigation of electronic transitions in alpha-helical and polyproline II-like conformations.

UV resonance Raman (UVRR) excitation profiles and Raman depolarization ratios were measured for a 21-residue predominantly alanine peptide, AAAAA(AAARA) 3A (AP), excited between 194 and 218 nm. Excitation within the pi-->pi* electronic transitions of the amide group results in UVRR spectra dominated by amide vibrations. The Raman cross sections and excitation profiles provide information about the nature of the electronic transitions of the alpha-helix and polyproline II (PPII)-like peptide conformations. AP is known to be predominantly alpha-helical at low temperatures and to take on a PPII helix-like conformation at high temperatures. The PPII-like and alpha-helix conformations show distinctly different Raman excitation profiles. The PPII-like conformation cross sections are approximately twice those of the alpha-helix. This is due to hypochromism that results from excitonic interactions between the NV 1 transition of one amide group with higher energy electronic transitions of other amide groups, which decreases the alpha-helical NV 1 (pi-->pi*) oscillator strengths. Excitation profiles of the alpha-helix and PPII-like conformations indicate that the highest signal-to-noise Raman spectra of alpha-helix and PPII-like conformations are obtained at excitation wavelengths of 194 and 198 nm, respectively. We also see evidence of at least two electronic transitions underlying the Raman excitation profiles of both the alpha-helical and the PPII-like conformations. In addition to the well-known approximately 190 nm pi-->pi* transitions, the Raman excitation profiles and Raman depolarization ratio measurements show features between 205-207 nm, which in the alpha-helix likely results from the parallel excitonic component. The PPII-like helix appears to also undergo excitonic splitting of its pi-->pi* transition which leads to a 207 nm feature.

Dependence of glycine CH2 stretching frequencies on conformation, ionization state, and hydrogen bonding.

We experimentally and theoretically examined the conformation, pH, and temperature dependence of the CH2 stretching frequencies of glycine (gly) in solution and in the crystalline state. To separate the effects of the amine and carboxyl groups on the CH2 stretching frequencies we examined the Raman spectra of 2,2,2-d3-ethylamine (CD3-CH2-NH2) and 3,3,3-d3-propionic acid (CD3-CH2-COOH) in D2O. The symmetric (nusCH2) and asymmetric (nuasCH2) stretching frequencies show a significant dependence on gly conformation. We quantified the relation between the frequency splitting (Delta = nuasCH2-nusCH2) and the xi angle which determines the gly conformational geometry. This relation allows us to determine the conformation of gly directly from the Raman spectral frequencies. We observe a large dependence of the nusCH2 and nuasCH2 frequencies on the ionization state of the amine group, which we demonstrate theoretically results from a negative hyperconjugation between the nitrogen lone pair and the C-H antibonding orbitals. The magnitude of this effect is maximized for C-H bonds trans to the nitrogen lone pair. In contrast, a small dependence of the CH2 stretching frequencies on the carboxyl group ionization state arises from delocalization of electron density from carboxyl oxygen to C-H bonding orbitals. According to our experimental observations and theoretical calculations the temperature dependence of the nusCH2 and nuasCH2 of gly is due to the change in the hydrogen-bonding strength of the amine/carboxyl groups to water.

UV Raman spatially resolved melting dynamics of isotopically labeled polyalanyl peptide: slow alpha-helix melting follows 3(10)-helices and pi-bulges premelting.

We used UV resonance Raman (UVRR) to examine the spatial dependence of the T-jump secondary structure relaxation of an isotopically labeled 21-residue mainly Ala peptide, AdP. The AdP penultimate Ala residues were perdeuterated, leaving the central residues hydrogenated, to allow separate monitoring of melting of the middle versus the end peptide bonds. For 5 to 30 degrees C T-jumps, the central peptide bonds show a approximately 2-fold slower relaxation time (189 +/- 31 ns) than do the exterior peptide bonds (97 +/- 15 ns). In contrast, for a 20 to 40 degrees C T-jump, the central peptide bond relaxation appears to be faster (56 +/- 6 ns) than that of the penultimate peptide bonds (131 +/- 46 ns). We show that, if the data are modeled as a two-state transition, we find that only exterior peptide bonds show anti-Arrhenius folding behavior; the middle peptide bonds show both normal Arrhenius-like folding and unfolding. This anti-Arrhenius behavior results from the involvement of pi-bulges/helices and 3(10)-helix states in the melting. The unusual temperature dependence of the (un)folding rates of the interior and exterior peptide bonds is due to the different relative (un)folding rates of 3(10)-helices, alpha-helices, and pi-bulges/helices. Pure alpha-helix unfolding rates are approximately 12-fold slower (approximately 1 micros) than that of pi-bulges and 3(10)-helices. In addition, we also find that the alpha-helix is most stable at the AdP N-terminus where eight consecutive Ala occur, whereas the three hydrophilic Arg located in the middle and at the C-terminus destabilize the alpha-helix in these regions and induce defects such as pi-bulges and 3(10)-helices.

Peptide secondary structure folding reaction coordinate: correlation between uv raman amide III frequency, Psi Ramachandran angle, and hydrogen bonding.

We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII3 frequency to its Psi Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Psi Ramachandran angle from measured UVRR AmIII3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Phi Ramachandran angle dependence and allow determination of the Psi angle with a standard error of +/-8 degrees , if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Psi angle by +/-6 degrees . The resulting ability to measure Psi spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Psi angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Psi reaction coordinate.

UV resonance Raman determination of polyproline II, extended 2.5(1)-helix, and beta-sheet Psi angle energy landscape in poly-L-lysine and poly-L-glutamic acid.

UV resonance Raman (UVR) spectroscopy was used to examine the solution conformation of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) in their non-alpha-helical states. UVR measurements indicate that PLL (at pH = 2) and PGA (at pH = 9) exist mainly in a mixture of polyproline II (PPII) and a novel left-handed 2.5(1)-helical conformation, which is an extended beta-strand-like conformation with Psi approximately +170 degrees and Phi approximately -130 degrees . Both of these conformations are highly exposed to water. The energies of these conformations are very similar. We see no evidence of any disordered "random coil" states. In addition, we find that a PLL and PGA mixture at neutral pH is approximately 60% beta-sheet and contains PPII and extended 2.5(1)-helix conformations. The beta-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Psi Ramachandran angles for these conformations, which we used to estimate a Psi Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes.